The plasma membrane of eukaryotic cells serves as a barrier against invading toxins, including certain bacteria. Endocytosis is the main transfer pathway of such items into immune system, barrier endothelial, and epithelial cells. Morphologically, endocytosis occurs by the formation of an invagination in the cell membrane, transformation of the invagination into a vesicle or continuous irregular channel, and its motion towards the other side or inside the cell. All nucleated interphase cells express continuous, high-capacity endocytotic activity wherein components of the cell medium are internalized in membrane-bound vesicles.
Receptor mediated endocytosis occurs primarily through clatrin-coated vesicles approximately 100 nanometers (nm) in diameter which form by the invagination of specialized coated-pit domains of the plasma membrane. Coated vesicles achieve efficient uptake of toxins, viruses, nutrient carriers, growth factors, peptide hormones, antigens, and other physiological ligands that bind to specific receptors expressed on the cell surface (see Cell, 32:663-667, 1983). In addition, solutes and particles are internalized nonspecifically in the fluid media of the vesicles by the process of fluid-phase endocytosis (see J. Cell Biol., 96:1-27, 1983).
The cells of the immune system are responsible for host reaction to the invasion of foreign materials into the body. Prevention and elimination of bacterial infection is a primary function of immune system cells, which is accomplished in part by endocytotic activity.
A particularly severe form of bacterial infection is sepsis, the generalized occupation of the body by bacteria. Sepsis and associated endotoxemia are among the most important pathologic situations where an unsatisfactory response of the immune system may lead to death. Presently, Gram-negative septicemia is among the leading causes of death in hospitals, despite potent antibiotics and vasoactive drugs. The fatality due to septicemia has remained 30 to 50% in the last decade.
Experimental evidence in both humans and animals has shown that many of the features of Gram-negative septicemia are due to endotoxin (see New Eng. J. Med., 307:1225-1230, 1982). Endotoxin is a lipopolysaccharide (LPS) present in the cell wall of certain bacterial species, including Gram-negative bacteria. The LPS molecule consists of a polysaccharide region covalently bound to a lipid region, termed lipid A. The lipid A molecules mediate most of the biological effects of endotoxins (see Am. J. Pathol., 93:527-617, 1978). Normal biological barriers are efficient to prevent endotoxin entry. In humans the gut contains large amounts of Gram-negative bacteria, that continuously project endotoxin. This endotoxin may transport across gut mucosa and then be removed by Kupffer cells of the liver (see Hepatology, 1:458-465, 1981).
Endotoxin induces many deleterious biological reactions in humans including fever, hypotension, shock and disseminated intravascular coagulation (see Ann. Rev. Med., 38:417-432, 1987). Endotoxin activates the coagulation, fibrinolytic and complement system (see Mol. Immunol., 24:319-332, 1987). Endotoxin also stimulates the arachidonic acid system, and activates polymorphonuclear cells, leukocytes and macrophages (see J. Clin. Invest., 77:1233-1243, 1986).
After contact with endotoxin, macrophages release special peptides, called cytokines. Among them are a few of a major importance: Interleukin-1 and Tumor Necrosis Factor (TNF) (see The Lancet, I:1122-1126, 1989). Cytokines modulate the development of alterations in cell function. All organs are the targets for endotoxin action in septicemia, however the lungs, liver and kidney are in particular damaged in sepsis, which leads to the development of respiratory, liver and kidney failure. Endotoxin induces cardiovascular disfunction and shock, which together lead to death.
Bacterial infection in sepsis is a subject to antibiotic therapy. However, the main unresolved problem in sepsis treatment remains endotoxemia, which requires, first of all, scavenging and inactivation of LPS. Attempts at treatment using monoclonal antibodies against LPS or LPS-binding protein have not proven an effective mode of therapy (see Discover, Nov:115-119, 1993). However, there are natural scavengers of LPS: The lipoproteins.
Endotoxemia is accompanied by alterations in lipid metabolism. Hyperlipidemia occurs in endotoxin-treated animals (see Am. J. Physiol., 253:E59-64, 1987), as well as during Gram-negative bacterial infection (see Clin. Chem., 32:142-145, 1986). In endotoxin-induced hyperlipidemia, a increased concentration of plasma triglycerides, very-low density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL), as well as decreased activity of lipoprotein-lipase (LPL), is observed. Also, activity of hepatic triglyceride-lipase is decreased both in experimental animals and humans (see Metabolism, 37:859-865, 1988). Hyperlipidemia results from accumulation of VLDL and LDL in plasma and delayed clearance of these lipoproteins, or increased synthesis of lipoproteins.
Lipoproteins are directly involved in host response to infection, and endotoxin-induced hyperlipidemia represents a physiological defence mechanism. Endotoxin interacts with cholesterol-ester rich lipoproteins. HDL have been shown to bind endotoxin (see J. Clin. Invest., 67:827-837, 1981). After binding to HDL, the ability of endotoxin to induce fever, leucocytosis and hypotension is dramatically reduced (see J. Clin. Invest., 62:1313-1324, 1978). In rabbits LDL can also bind endotoxin in the same way and LDL-bound endotoxin shows less toxicity. It has also been shown (see J. Clin. Invest., 86:696-702, 1990) that VLDL, chylomicrons and artificial lipid emulsions can protect mice against endotoxin-induced death. Furthermore, triglyceride-rich lipoproteins inactivate endotoxin in vitro. Triglyceride-rich lipid solutions and chylomicrons have been shown to significantly improve survival in animals with sepsis and reduce the serum level of endotoxin and TNF (see J. Clin. Invest., 91:1028-1034, 1993).
As noted above, the entry step for many antigens, such as bacteria, into cells is determined by membrane-related processes, such as endocytosis. These processes include changes in membrane shape and conformation, and thus may be affected by physico-chemical factors.
There are several known approaches that disrupt receptor-mediated endocytosis or non-receptor mediated endocytosis and phagocytosis, and membrane trafficking. They involve depletion of calcium or other ions, application of cytochalasin or related substances to prevent actin polymerization in cells, and inhibition of the cell energy production by toxins. However, all these approaches are not applicable for the purposes of treatment of a whole living organism to combat topical bacterial infection.
In treatment of sepsis, application of specific antibodies to particular receptors or antigens was not shown to be beneficial in clinical practice. Usually the exact type of antigen in disease is not known. The immense variety of antigens makes the attempts to use specific antibodies impractical in wide medical practice. Also, immunotherapy in these cases is extremely expensive. Other approaches to inactivate endotoxins and to treat septic shock include the use of peroxy-diphosphate compounds (U.S. Pat. No. 5,034,383), steroids (U.S. Pat. No. 4,844,894) and lipid analogues-sphingosine (U.S. SIR No. H1,168).
Previous studies have shown that detergents are strong modulators of membrane-related processes (see Attwood D. & Florence A., Surfactant Systems (1983)). It is well known that detergents and related pharmaceutical drugs at appropriate concentrations stabilize biological membranes and attenuate, for example, erythrocyte osmotic lysis. Detergents are also reported to be successfully used in treating vascular and is chemic disorders (U.S. Pat. Nos. 5,152,979, 5,240,702, 5,080,894, 5,078,995, 5,089,260 and 5,182,106).
Detergents of different origin are important components of all body liquids. Interaction between detergents, lipids and proteins determines cell membranes fluidity, permeability and activity of membrane enzymes. Proteins themselves are evidently the most important and potent surfactants of the body. Bile salts are also important native surfactants, as well as corticosteroids, polypeptides and lipoproteins.
Phagocytosis of bacteria by neutrophils is dependant upon the adhesiveness of bacteria and neutrophils, which is surface related phenomena (see van Oss, C. J. et al., Phagocytic Engulfment and Cell Adhesiveness (1975)). Studies have shown that application of detergents sodium deoxycholate and Tween 80 at 0.01% to the cell media reduced phagocytosis of Staphylococcus epidermis by human neutrophils by four-fold. Detergents oppose endocytosis and promote exocytosis. Antibiotics of the polymyxin group, which are polycationic detergents of a polypeptide nature, interfere with the bacterial membranes, causing appearance of numerous profusions or blebs, extending from the outside surface of the cell. The same processes were also observed in non-bacterial cells (see Storm, D. L. et al., Polymyxin and related peptide antibiotics, Ann. Rev. Biochem., 46:723-763 (1977).
In addition, in vitro studies have shown that synthetic phosphatidylcholine stopped vesiculation in platelets, and at an appropriate concentration induced the opposite process--formation of cell podii and zeiosis (see J. Pharmacobio-Dynamics, 9:131-137, 1986). Endocytosis of horseradish peroxidase in smooth muscle and endothelial cell cultures was inhibited in dose and time dependant manner by cholestane and hydroxycholesterol (see Artery, 17:84-95, 1990). In vitro adherence and migration of granulocytes were inhibited by Pluronic F68. The same effect was also demonstrated in vivo (see Proc. Soc. Exp. Biol. Med., 188:461-470, 1988).
Detergents can provide a direct lytic action on bacterial cells in vitro. Detergents are also known to induce hyperlipidemia. Tyloxapol (e.g., Triton.RTM. WR-1339) is the most widely used detergent to induce hyperlipidemia in animal models (see J. Exp. Med., 114:279-293, 1961). Lipidemia and increase in plasma LDL has been reported after infusion of Triton.RTM. A-25, Polysorbate-80, Pluronic and Cremophor EL (see Attwood, et al., 1980). The mechanisms involved are blockade of lipoprotein-lipase and LDL receptors, and formation of "micelies" with lipoproteins.
As described above, plasma lipoproteins possess a marked anti-endotoxin effect, and detergents have a regulatory effect on membrane-related transfer processes in cells. However, the use of biologically active detergents for modification of lipid metabolism and plasma levels of lipoproteins and membrane traffic modification for the treatment of endotoxemia and sepsis has not been previously disclosed.
Accordingly, it is an object of the present invention to provide such a method, useful in the treatment of bacterial infection and associated toxemia, based on the physico-chemical properties of non-ionic detergents.